Thin-layer chromatographic determination of erythromycin and other macrolide antibiotics in livestock products

A method is described for determination of 4 macrolide antibiotics in livestock products. Erythromycin, tylosin, oleandomycin, and spiramycin were extracted from animal tissues, milk, and egg with acetonitrile at pH 8.5. Cleanup was done by adding sodium chloride and dichloromethane, evaporating the organic layer, and subsequent acid/base partitioning. After the antibiotics were separated by thin-layer chromatography (TLC), they were reacted with xanthydrol and could be detected as purple spots down to 0.02 mg/kg without interference by other commonly used therapeutic drugs (23 were tested). Anisaldehyde-sulfuric acid, cerium sulfate-molybdic acid, phosphomolybdic acid, and Dragendorff's reagent proved to be less sensitive as visualizing agents. For quantitation, TLC plates were scanned at 525 nm. Recoveries were between 71 and 96% for erythromycin and tylosin in liver, muscle, and egg at the 0.1-0.5 mg/kg level and 51% for erythromycin in milk at the 0.02 mg/kg level (coefficient of variation = 10-18%). Bioautography with Bacillus subtilis was used to confirm results, in addition to TLC analysis of derivatized antibiotics and liquid chromatography with electrochemical detection. Various derivatization procedures for erythromycin were investigated for improved ultra-violet or fluorescence detection in liquid chromatography.     

Thin layer chromatographic determination of citrinin.

A thin layer chromatographic (TLC) method is described for the determination of citrinin in feeds. Citrinin is extracted from feed with methanol and water, the mixture is made alkaline with 10% sodium carbonate, and the aqueous solution is filtered and extracted with chloroform to remove most of the interfering materials. The aqueous layer is acidified with 2N HCl and extracted with chloroform. The chloroform extract is concentrated and spotted on a thin layer chromatographic (TLC) plate which is developed in chloroform-acetone-ethanol-water (60 + 40 + 10 + 1). The citrinin is viewed under ultraviolet light after TLC. Either visual or fluorodensitometric quantitation is used. Recoveries of citrinin from various feed samples spiked at levels of 2.0--5 micrograms/g were 75--92%. The proposed method can detect 0.5 micrograms/g feed, including corn, silage, ready mixed feeds, and feed pellets.     

Dilution errors in aflatoxin determinations caused by compounds extracted from peanuts

Several methods have been developed to analyze peanuts for aflatoxin by using thin layer chromatography (TLC). These methods depend on solvent extraction of aflatoxin from a sample of the product. Unfortunately, solvent solutions used to extract aflatoxin from peanuts also extract measurable quantities of other compounds such as oils, fats, sugars, and protein. The volume of these extracted compounds causes error in measuring the proportion of the solvent solution analyzed for aflatoxin. Also, because the cleanup procedures for some methods are inadequate, the volume of some of these extracted compounds also causes error in measuring the proportion of the extracted aflatoxin placed on TLC plates. These 2 errors cause underestimation of aflatoxin concentrations by approximately 11, 14, and 5% for the CB method, the modified version of the BF method generally used for raw peanuts, and a water slurry method, respectively. The correction specified by the CB method for fats in the extraction solvent reduces the approximate error for the CB method from 11 to 1%.     

Determination of polybrominated biphenyl residues in dairy products.

A method for the determination of polybrominated biphenyls (PBBs) in dairy products is described. Fat is extracted from the products by the official AOAC method. The PBB residues are separated from the fatty material by gel permeation chromatography prior to gas-liquid chromatographic (GLC) quantitation. An additional cleanup using petroleum ether elution through a miniature Florisil column is necessary for thin layer chromatographic (TLC) confirmation. Recoveries of PBBs from samples fortified at levels from 0.1 to 0.5 ppm ranged from 94 to 104% with an average of 99%. GLC sensitivity permits the estimation of PBB residue levels as low as 0.007 ppm. Routine TLC confirmation is limited by sensitivity to greater than or equal to 0.2 ppm.     

Rapid thin layer chromatographic method for determining aflatoxin B1, ochratoxin A, and zearalenone in corn

A multimycotoxin thin layer chromatographic method is described for the analysis of corn. Aflatoxins are extracted from the samples with acetonitrile-water, and sodium bicarbonate is added to separate the acidic ochratoxin from zearalenone and aflatoxin B1. After chloroform extraction, 1N NaOH is added to separate zearalenone and aflatoxin B1. The separated mycotoxins are spotted on TLC plates, which are then examined under ultraviolet light. The following recoveries (%) were obtained for corn samples: aflatoxin B1 71, ochratoxin A 87, and zearalenone 85. The limits of detection for the respective mycotoxins were 2, 40, and 200 ppb.     

Thin layer chromatography and densitometric determination of aflatoxins in mixed feeds containing citrus pulp

A method for the accurate one-dimensional thin layer chromatographic (TLC) determination of aflatoxins B1, B2, G1, and G2 in mixed feeds is presented. The aflatoxins are extracted from the sample with chloroform and purified by solvent partitioning. Each aflatoxin is separated from pulp interference by thin layer chromatography on aluminum-backed silica plates. The separated aflatoxins are detected by fluorescence densitometry. Average recoveries for samples spiked from 10 to 100 ppb B1 and G1 and from 3 to 30 ppb B2 and G2 are 82, 84, 95, and 94% for B1, B2, G1, and G2, respectively. The above recovery data, when analyzed for overall method repeatability, produced relative standard deviations of 6.8, 4.3, 6.9, and 7.6% for B1, B2, G1, and G2, respectively. Minimum detection level is less than 1 ppb for each aflatoxin. B1 is confirmed by trifluoroacetic acid derivative formation on a silica TLC plate.     

Rapid quantitation and confirmation of aflatoxins in corn and peanut butter, using a disposable silica gel column, thin layer chromatography, and gas chromatography/mass spectrometry

A simple, rapid, and solvent-efficient method for determining aflatoxins in corn and peanut butter is described. Aflatoxins B1, B2, G1, and G2 were extracted from 50 g sample with 200 mL methanol-water (85 + 15). A portion of the extract was diluted with 10% NaCl solution to a final concentration of 50% methanol, and then defatted with hexane. The aflatoxins were partitioned into chloroform. The chloroform solution was evaporated, and the residue was placed on a 0.5 g disposable silica gel column. The column was washed with 3 mL each of hexane, ethyl ether, and methylene chloride. Aflatoxins were eluted with 6 mL chloroform-acetone (9 + 1). The solvent was removed by evaporation on a steam bath, and the aflatoxins were determined using thin layer chromatography (TLC) with silica gel plates and a chloroform-acetone (9 + 1) developing solvent. Overall average recovery of aflatoxin B1 from corn was 82%, and the limit of determination was 2 ng/g. For mass spectrometric (MS) confirmation, aflatoxin B1 in the extract from 3 g sample (20 ng/g) was purified by TLC and applied by direct on-column injection at 40 degrees C into a 6 m fused silica capillary gas chromatographic column. The column was connected directly to the ion source. After injection, the temperature was rapidly raised to 250 degrees C, and the purified extract was analyzed by negative ion chemical ionization MS.     

Antioxidant activity of extracts obtained from residues of different oilseeds

Residues of the oil-extracting process of oilseeds contain bioactive substances such as phenolic compounds, which could be used as natural antioxidants for the protection of fats and oils against oxidative deterioration. Thus, the extraction of such constituents from residual material can be considered to contribute to the added value of these residues, which could justify their isolation. In the present work the fat-free residues of eight different oilseeds whose oils are usable for nutritional applications, and also as renewable resources, were extracted with 70% methanol, 70% acetone, water, and ethyl acetate/water, respectively. The resulting extracts were investigated regarding their content of total phenolic compounds by the Folin-Ciocalteau assay, sinapine, flavanoids, and the UV-absorption spectra. Further, the antioxidant activity of the extracts was characterized by the DPPH method, the beta-carotene-linoleic acid assay, and ESR investigations. The fat-free residues of the different oilseeds contained considerable amounts of extractable substances. The yields decreased with decreasing polarity of the solvent in the order water, 70% methanol, 70% acetone, and ethyl acetate. The ratio of total phenolic compounds to the extractable compounds ranged from 3 to 19%. There was no significant correlation between the amount of total extractable compounds and the total phenolic compounds (p < 0.001). All extracts showed remarkable antioxidant activities determined with the different methods. The effects depended strongly on the solvent used for the extraction as well as on the extracted residue. A correlation between the methods used for the characterization of the antioxidant activity and the composition of the residues could not be shown.     

Microbiological determination of penicillin G, ampicillin, and cloxacillin residues in milk

A fast cylinder plate microbiological method was developed for the quantitative determination of penicillin G, ampicillin, and cloxacillin in milk. Agar plates seeded with stable spores of Bacillus stearothermophilus var. calidolactis were used and incubated at 64 degrees C for 4 1/2 hr. Standard curves were obtained for the following ranges of concentration of antibiotics: 0.004-0.064 IU penicillin G/mL, 0.0025-0.04 microgram ampicillin/mL, and 0.03-0.48 microgram cloxacillin/mL. The method is suitable for detecting penicillin residues in milk and for quantitative milk-out studies of the above antibiotics used in treatment of bovine mastitis.     

Chemical confirmatory tests for ochratoxin A, citrinin, penicillic acid, sterigmatocystin, and zearalenone performed directly on thin layer chromatographic plates

Published tests have been improved and a new procedure is described for chemical confirmation of mycotoxins directly on thin layer plates. After extraction and preliminary cleanup chromatography with n-hexane or chloroform, the mycotoxins ochratoxin A, citrinin, penicillic acid, sterigmatocystin, and zearalenone were easily separated by thin layer chromatography (TLC) using toluene-ethyl acetate-90% formic acid (6 + 3 + 1) developing solvent. In chemical confirmatory methods, the developed chromatogram was exposed to vapors of pyridine, acetic anhydride, or a mixture, or the mycotoxins were over-spotted. With this treatment, ochratoxin A, citrinin, penicillic acid, and zearalenone were converted to new fluorescent compounds, and observed under 365 nm light after re-chromatography with the same developing solvent. Sterigmatocystin was confirmed chemically using TLC plates impregnated with 0.6N H2SO4 or 10% oxalic acid in methanol. The described procedures are satisfactory for confirming mycotoxins present in standards, artificially contaminated grain samples (barley, corn, oat, rye, and wheat), and extracts from both fungal cultures and naturally contaminated grain samples.     

Determination of polysorbates in foods by colorimetry with confirmation by infrared spectrophotometry, thin-layer chromatography, and gas chromatography

A method is presented for the detection of polysorbates (PSs) in 8 kinds of processed foods by colorimetric and thin-layer chromatographic (TLC) techniques. The PSs are extracted from processed foods with a mixture of methylene chloride and ethanol by using an Extrelut column. The extract is further purified by using a silica gel column. The PS extract is complexed with cobalt-thiocyanate (Cothiocyanate) reagent and is determined spectrophotometrically at 620 nm. The recoveries and coefficients of variation for 8 kinds of processed foods fortified with 0.1% PS 80 were 67.9-94.6% and 4.0-11.3%, respectively. The detection limit of TLC corresponded to 50 mg PS 80/kg. PS identity was confirmed by infrared spectrophotometry of PS extract, and gas chromatography of fatty acids and thin layer chromatography of POE-sorbitan residues after saponification.     

A screening method for sulfonamides extracted from animal tissues.

A screening method for the estimation of possible residues of sulfonamides in poultry tissues is described. The method utilizes thin layer chromatography (TLC) to separate Bratton-Marshall positive reactants. In the absence of interference and the identification of 1 sulfonamide by TLC, the colorimetric method is recommended for quantitation. When interferences are present, TLC should be used for both qualitative and quantitative analysis. The screening method has a sensitivity of less than 0.05 ppm and a recovery of greater than 80%.     

Screening and mass spectral confirmation of beta-lactam antibiotic residues in milk using LC-MS/MS.

Milk is typically screened for beta-lactam antibiotics by nonspecific methods. Although these methods are rapid and sensitive, they are not quantitative and can yield false positive findings. A sensitive and specific method for the quantitation and mass spectral confirmation of five beta-lactam and two cephalosporin antibiotics commonly or potentially used in the dairy industry is described using high-performance liquid chromatography with tandem mass spectrometry. The antibiotics studied were ampicillin, amoxicillin, penicillin G, penicillin V, cloxacillin, cephapirin, and ceftiofur. The antibiotics were extracted from milk with acetonitrile, followed by reversed-phase column cleanup. The extract was analyzed by liquid chromatography coupled with a mass spectrometer, using a water/methanol gradient containing 1% acetic acid on a C-18 reversed-phase column. Determination was by positive ion electrospray ionization and ion trap tandem mass spectrometry. Quantitation was based on the most abundant product ions from fragmentation of the protonated ion for amoxicillin, cephapirin, ampicillin, and ceftiofur and on the fragmentation of the sodium adduct for penicillin G, penicillin V, and cloxacillin. The method was validated at the U.S. FDA tolerance or safe level and at 5 or 2.5 ng/mL for these compounds in bovine milk. Theoretical method detection limits in milk based on a 10:1 signal to noise ratio were 0.2 ng/mL (ampicillin), 0.4 ng/mL (ceftiofur), 0.8 ng/mL (cephapirin), 1 ng/mL (amoxicillin and penicillin G), and 2 ng/mL (cloxacillin and penicillin V) using a nominal sample size of 5 mL.     

Solvent extraction characterization of bioavailability of atrazine residues in soils

Characterization of pesticide bioavailability, particularly in aged soils, is of continued interest because this information is necessary for environmental risk assessment. The objective of this study was to correlate atrazine residue bioavailability in aged soils, as determined by solvent extraction methods, to atrazine mineralization by an atrazine-degrading bacterium. Webster clay loam and Zimmerman fine sand soils were treated with UL-ring-labeled [14C]atrazine and incubated for up to 8 weeks. At the end of each incubation period, soils were either not extracted, extracted with 0.01 M CaCl2, or extracted with 0.01 M CaCl2/aqueous methanol. Soils were then inoculated with the bacterium Pseudomonas sp. strain ADP, which is capable of rapidly mineralizing the atrazine ring. This allowed for the evaluation of the bioavailability of aged atrazine residues without the contribution of atrazine desorption from soil. Results of these studies indicated that the amounts of atrazine residues in aged soils extracted by 0.01 M CaCl2 and aqueous methanol were correlated to amounts of atrazine mineralized by Pseudomonas sp. strain ADP. Consequently, 0.01 M CaCl2/methanol extractable atrazine in aged soils may be used to estimate bioavailable residues, and this technique may be useful to determine the bioavailability of other compounds in soils, especially other triazine herbicides.     

Ion-pairing liquid chromatographic determination of benzimidazole fungicides in foods

A method is described for determining residues in foods of thiabendazole, thiophanate methyl, the di-oxygen analogue metabolite [dimethyl 4,4'-O-phenylene bis (allophanate)] that is the metabolite name of the latter, and methyl-2-benzimidazole carbamate, which is the major metabolite and fungitoxic principle common to both thiophanate methyl and benomyl. The residues are extracted from the product using methanol and are partitioned into dichloromethane after initial acidification and again after subsequent alkalinization of the extract. Residues are separated and quantified by reverse-phase liquid chromatography using an ion-pairing mobile phase with UV and fluorescence detectors in tandem. Recoveries from 7 different food crops fortified at 0.2-35 ppm levels ranged from 64 to 105%.     

Antibiotic identification by high voltage electrophoresis bioautography

The high voltage electrophoresis bioautography method is applicable to meat, milk, and animal feeds. Meat is freeze-dried, powdered, and extracted with acetonitrile-water (9 + 1), and the extract is concentrated by evaporation at room temperature. Milk is examined directly or following acetonitrile-water extraction. Feed is extracted with acetonitrile-water. Samples or extracts are applied to preliminary assay plates of antibiotic medium No. 1 at pH 6 and 8, seeded with Micrococcus luteus (ATCC 9341), M. luteus DHSR (ATCC 9341A), Bacillus cereus (ATCC 11778), or B. cereus K250 TR (NCIB 11183), and nutrient agar at pH 7 seeded with B. subtilis BGA. Inactivation of penicillinase indicates beta-lactam antibiotics. Addition of trimethoprim increases sensitivity to sulfonamides. After 18-24 h incubation at 30 degrees C, plates yielding clear inhibition zones guide selection of conditions for subsequent electrophoresis bioautography. Extracts are applied (5-100 microL) to 10 mm diameter wells on electrophoresis plates 60 cm long and 40 cm wide, with a gel depth of 1.6 mm. The support medium is 1% agar and 1% agarose in Tris/succinic acid buffers pH 6 and pH 8. A potential of 1500 V is applied for 1.5 h at 15 degrees C. Following electrophoresis, the migrated antibiotics are visualized by over-layering with antibiotic medium No. 1, pH 6 or 8, seeded with M. luteus or B. cereus spore suspension; plates are incubated for 18-24 h at 30 degrees C. Identification is based on results of preliminary screening together with electrophoretic migration distances and inhibition zone appearances compared with standards.     

Improved cleanup for gas chromatographic determination of propiconazole residues in soil, wheat grain, straw, and leaves

A simple and sensitive method is described for determination of propiconazole, a new type of broad-spectrum systemic fungicide, in soil, wheat grain, straw, and leaves. Pesticide residues in or on grain and green plant materials are extracted with methanol (or a mixture of methanol and water (4 + 1), for soil), partitioned into methylene chloride, and cleaned up on an alumina column for grain and soil or an activated charcoal column for green plant materials. The amount of residue is quantitatively measured by gas chromatography using an alkali flame ionization detector in the nitrogen-sensitive mode. Recoveries from soil, grain, and green plant materials fortified at 0.1-5 mg/kg are better than 80%. The practical detection limits of this method are 0.01 mg/kg in grain and soil and 0.02 mg/kg in green plant materials.     

Influence of soil aging on sorption and bioavailability of simazine

Characterization of pesticide bioavailability, particularly in aged soils, is of continued interest because this information is necessary for environmental risk assessment. However, pesticide bioavailability in aged soils has been characterized by a variety of methods with limited success, due in part to methodological limitations. The objective of this study was to use solvent extraction methods to correlate simazine residue bioavailability in aged soils to simazine mineralization using a simazine-mineralizing bacterium. Soils from Brazil, Hawaii, and the midwestern United States were treated with UL-ring-labeled [14C]simazine and incubated for up to 8 weeks. At the end of each incubation period, soils were either incubated further, extracted with 0.01 M CaCl2, or extracted with aqueous methanol (80:20 v/v methanol/water). In a parallel experiment, after each incubation period, soils were inoculated with the bacterium Pseudomonas sp. strain ADP, which is capable of rapidly mineralizing simazine, and 14CO2 was determined. The inoculated soil samples were then extracted with 0.01 N CaCl2 and with aqueous methanol. This allowed for the evaluation of the bioavailability of aged simazine residues, without the contribution of simazine desorption from soil. Results of these studies indicated that simazine sorption to soil increased with aging and that amounts of simazine in aged soils extracted by 0.01 M CaCl2 and aqueous methanol were highly correlated to amounts of simazine mineralized by Pseudomonas sp. strain ADP. Consequently, 0.01 M CaCl2/methanol-extractable simazine in aged soils can be used to estimate bioavailable residues. This technique may be useful in determining the bioavailability of other s-triazine compounds in soils.     

Immunochromatography of fusarochromanone mycotoxins

The present paper describes an enzyme-linked immunoassay (ELISA) used in combination with thin-layer chromatography (TLC) and liquid chromatography (LC) for determination of fusarochromanone (TDP) mycotoxins in barley, wheat, and a Fusarium culture grown in rice and corn. The mycotoxins were first extracted from the sample with 100% methanol and subjected to TLC or LC without additional cleanup treatment. Individual fractions eluted from TLC or LC were acetylated, then analyzed by ELISA. Determinations of TDP toxins at levels as low as 0.1 and 0.5 ng were achieved by ELISA in combination with LC and TLC, respectively. The detection limit for TDP-1 in barley and wheat was about 20 ppb by ELISA alone as compared with a detection limit of 5 ppb by a combination of ELISA with either TLC or LC. Overall analytical recovery (% of added) of TDP-1 added to barley and wheat at 5, 10, and 20 ppb of TDP-1 was 106.9 +/- 15.3 and 113.2 +/- 11.6 by LC-ELISA and 108.8 +/- 9.1 and 110.4 +/- 4.9 by TLC-ELISA, respectively. Analysis of extracts obtained from Fusarium equiseti R6137 grown in corn and rice by the combination of TLC and ELISA revealed that diacetyl-TDP was also produced by this fungus in addition to TDP-1 and TDP-2. Comparable results were obtained when fungal extracts were subjected to ELISA, LC, and immunochromatography (i.e., combination of ELISA with either TLC or LC).     

Electron capture gas-liquid chromatographic method for the simultaneous analysis of 2,4-D, dicamba, and mecoprop residues in soil, wheat, and barley.

A method has been developed for the simultaneous analysis of 2,4-D (2,4-dichlorophenoxy-acetic acid), dicamba (2-methoxy-3,6-dichloro-benzoic acid), and mecoprop (MCPP; 2-[(4-chloro-o-tolyl) oxy] propionic acid) residues in soil, wheat, and barley. Soil and crop samples are extracted with acidic acetone and methanol, respectively. The extracts in diethyl ether are esterified with diazomethane and cleaned up by passing through a Florisil column. Extracts are analyzed by gas-liquid chromatography, using an electron capture detector to determine 2,4-D and dicamba residues. Mecoprop in the extract is not detected at low levels of concentration. However, bromination of the extract increases the response of the electron capture detector to mecoprop. The method is sensitive to about 0.05 ppm 2,4-D and dicamba and 0.5 ppm mecoprop. Recoveries of these 3 herbicides added to soil, wheat, and barley samples at 0.05, 0.1, 0.5, and 1.0 ppm levels were between 65 and 93%. The method was used for the simultaneous analysis of 2,4-D, dicamba, and mecoprop residues in wheat, barley, and soil samples obtained from fields sprayed with the herbicide formulation Kil-Mor.